JP6961090B2 - Anti-PD-1 / CD47 bispecific antibody and its application - Google Patents

Description

The present disclosure relates to the fields of immunology and antibody engineering techniques, in particular bispecific antibodies against PD-1 and CD47, and their applications. More specifically, the present disclosure relates to recombinant antibodies, nucleic acids, constructs, methods of preparing recombinant antibodies, therapeutic compositions for treating cancer, and methods for treating cancer.

Cancer has become one of the leading causes of death worldwide in recent years. According to a report by the World Health Organization, the number of deaths from cancer worldwide in 2015 reached about 8.8 million, accounting for about one-sixth of the total number of deaths per year. In 2015, there were approximately 4,292,000 newly diagnosed cases of cancer in China and 2,814,000 deaths from cancer. In China, the incidence of cancer is still rising.

Currently, cancer treatments are primarily focused on surgery, radiation therapy, and chemotherapy, but conventional treatments have the disadvantages of low tumor specificity, high toxicity and side effects. Therefore, there is a strong need for safer and more effective methods with higher specificity for treating cancer. In recent years, as the understanding of the mechanism of cancer has deepened, molecular-targeted therapies that use specific antibodies to inhibit tumor growth are gradually appearing. Under these circumstances, antibody drugs prepared mainly by antibody engineering techniques based on cell engineering techniques and genetic engineering techniques show advantages such as high specificity, remarkable therapeutic effect, low toxicity and few side effects. It has a very wide potential for cancer treatment. Recently, the discovery of immune checkpoints has led to the development of specific antibodies that target immune checkpoints, blocking the immune escape of tumor cells and taking immunotherapy to a new level.

The immune system relies not only on the first stimulatory signal provided by the MHC-TCR complex, but also on the second stimulatory signal (ie, co-stimulatory signal) provided by molecules on the surface of antigen-presenting cells. , T cell activation is complicated. In the absence of such co-stimulation signals, it causes T cell non-responsiveness, tolerance and apoptosis. Programmed death receptor-1 (also known as PD-1, CD279) and its ligands (PD-1 ligand 1 and ligand 2, ie PD-L1 and PD-L2) are thus described. Inhibitive costimulatory molecule.

Human PD-1 belongs to the CD28 family of type I transmembrane glycoproteins and has a molecular weight of approximately 55 kDa. Human PD-1 contains two tyrosine residues in the intracellular domain (ie, Y223 and Y248), the N-terminal immunoreceptor tyrosine system inhibitory motif (ITIM, motif VDYGEL) and the C-terminal immunoreceptor tyrosine system, respectively. Involved in the formation of switch motifs (ITSM, motif TEYATI). The ITIM motif recruits intracellular SHP-2, phosphorylates downstream proteins, reduces BCR or TCR receptor signaling, and ultimately suppresses lymphocyte proliferation, cytokine production, and the cell division cycle. Induces a stoppage of. Human PD-1 has an IgV-like domain in the extracellular space. Such an IgV-like domain contains a plurality of sugar chain addition sites, is highly glycosylated, is mainly involved in binding to a ligand, and exerts a function of suppressing T cell activation. Unlike other costimulatory molecules that exist in dimeric form, PD-1 can exist as a monomer because the extracellular region of PD-1 lacks a cysteine residue. PD-1 is expressed on activated T cells and B cells, as well as monocytes, dendritic cells (DCs) and regulatory T cells (Tregs).

The two ligands for PD-1 (ie, PD-L1, B7-H1, CD274, and also known as PD-L2, B7-DC) are type I glycoproteins that belong to the members of the B7 protein family. The major ligands of PD-1 and PD-L1 include an IgV-like region, an IgC-like region, a transmembrane region, and a cytoplasmic region, which are involved in intracellular signal transduction, and the IgV-like region and the above. The IgC-like region is involved in cell-cell signaling. Expression of PD-1 is actively regulated by several inflammatory factors such as interleukin 4 (IL-4), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ). In addition to expression in activated T cells, B cells, macrophages, dendritic cells, and various tumor cells, PD-L1 is a non-lymphatic component of the heart, blood vessels, placenta, skeletal muscle, lungs, liver, spleen, thymus, etc. It is also widely expressed in systemic organs. PD-L2 is predominantly expressed in activated macrophages, DCs, and few tumor cells.

The immunosuppressive effect of the PD-1 / PD-L1 signaling pathway plays an important role in the development and development of various immune disorders, such as autoimmune diseases. Mice lacking PD-1 exhibit delayed organ-specific autoimmunity. The lack of PD-1 exhibits accelerated tissue-specific autoimmune properties in the lupus erythematosus LPR mouse model and the diabetic NOD mouse model including autoimmune disease. Furthermore, dysfunctional cytotoxic lymphocytes (CTL) that overexpress PD-1 are found in chronic infections caused by viruses such as HIV and HBV, whereas blocking PD-1 signals reverse the function of CTL. And remove the virus.

Tumor cells perform antigenic escape by immunosuppression of the PD-1 / PD-L1 signaling pathway. Various tumor cells such as non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, leukemia, various urinary tract tumors, gastrointestinal tumors, and genital system tumors can upregulate PD-L1 expression. Overexpressed PD-L1 interacts with PD-1 receptors on the surface of T cells, thereby phosphorylating tyrosine in the ITSM domain of PD-1 and downstream phosphatidylinositol 3-kinase (PI3K). And tyrosine kinase (Syk) dephosphorylation, thereby inhibiting the activation of downstream AKT, ERK, and other pathways, and ultimately the genes and cytokines required for T cell activation. Inhibits transcription and translation of. On the other hand, studies have shown that PD-L1 causes the accumulation of PD-1 in T cells, resulting in cell cycle arrest and massive accumulation of cells in the G0 / G1 phase. Activation of the PD-1 / PD-L1 signaling pathway can lead to apoptosis of certain CTLs, thereby desensitizing the cytotoxic killing effect of CTLs and promoting immune escape of tumor cells. However, it is also found by in vitro experiments and mouse models. In summary, tumor cells can inhibit T cell activation, proliferation and tumor killing ability by expressing PD-L1 that interacts with PD-1 on the surface of T cells.

Therefore, the PD-1 / PD-L1 signaling pathway, which plays an important role in tumor immunity, has become a new molecular target in tumor immunotherapy. Inhibition of T cells (ie, major cell-mediated immune cells) by tumor cells can be blocked by blocking the PD-1 / PD-L1 signaling pathway, anti-PD-1 antibodies and anti-PD-L1 The development of antibodies has become a general research direction in the study of tumor immunotherapy. Monoclonal antibodies that target PD-1 approved by the US Food and Drug Administration (FDA) include Keitruda (pembrolizumab) from Merck and Opdivo (nivolumab) from Bristol-Myers Squibb. Monoclonal antibodies that target PD-L1 include Tecentriq (atezolizumab) from Roche, Bavencio (avelumab) manufactured by Pfizer and Merck, and Imfinzi (durvalumab) from AstraZeneca. These monoclonal antibodies are used not only in melanoma and metastatic flat epithelial non-small cell lung cancer, and in other tumors that have progressed after chemotherapy (ie, the cancer to which the monoclonal antibody was first applied), but also in newly applied hodgkin. Good clinical results have already been achieved in lymphoma, renal cancer, gastric cancer, anal cancer, liver cancer, colorectal cancer and the like. In addition, several anti-PD-1 and anti-PD-L1 antibodies are in clinical trials for the treatment of tumors.

Currently, the greatest limitation of immunotherapy is low efficacy, such as 15% to 50% efficacy against PD-1. Evidence from the study shows that successful cancer immunotherapy depends not only on eliciting a local immune response of the tumor, but also on its ability to elicit a systemic immune response. Generating a systemic immune response when stimulating the immune system is expected to significantly improve the effectiveness of immunotherapy.

The present disclosure is intended to solve at least some of the technical problems in related technology.

In one embodiment, recombinant antibodies ( ie, recombinant proteins ) are provided in the present disclosure. In the embodiments of the present disclosure, the recombinant antibody comprises an anti-PD-1 antibody and an extracellular segment of human signaling protein α (SIRPA), and the N-terminus of the human SIRPA extracellular segment is the anti-PD-1 antibody. It is connected to the C-terminus of the heavy chain of the PD-1 antibody. Since the recombinant antibody according to this embodiment can target both PD-1 and CD47, it significantly enhances stimulation of the immune system and more potent tumor suppression than an antibody against a single target. You can demonstrate your abilities.

In embodiments of the present disclosure, the recombinant antibody can further comprise at least one of the following additional technical features:

In the embodiments of the present disclosure, the anti-PD-1 antibody is an IgG-like antibody against PD-1. Preferably, the IgG-like antibody is an IgG4 subtype. IgG4 easily forms incomplete antibodies by Fab-arm exchange. S228P modification of IgG4 subtype antibody can effectively reduce Fab arm exchange and effectively avoid the effects of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC).

In embodiments of the present disclosure, the anti-PD-1 antibody is an HX008 18A10 antibody (ie, also referred to herein as an H8 antibody) and is described in Patent Application 201610207741.6 and PCT / CN2016 / 103814. Refer to the contents.

In embodiments of the present disclosure, the recombinant antibody further comprises a linker, the N-terminus of the linker is connected to the C-terminus of the heavy chain of the anti-PD-1 antibody, and the C-terminus of the linker is said. It is connected to the N-terminus of the human SIRPA extracellular segment, avoids the interactive effects of protein steric disorders, and further promotes protein folding.

In embodiments of the present disclosure, the linker comprises the amino acid sequence of SEQ ID NO: 1.

In a second aspect, recombinant antibodies are provided in the present disclosure. In embodiments of the present disclosure, the recombinant antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2 ( ie, the light chain of an anti-PD1 antibody ) and a heavy chain comprising the amino acid sequence of SEQ ID NO: 3.

According to the first aspect and the disclosure in the present embodiment, the anti-PD-1 antibody of the recombinant antibody has the amino acid sequence of SEQ ID NO: 6 contained in the amino acid sequence of SEQ ID NO: 3 described above. It is shown to contain heavy chains that contain.

According to the first aspect and the disclosure in the present embodiment, the human SIRPA extracellular segment of the recombinant antibody has the amino acid sequence of SEQ ID NO: 7 contained in the amino acid sequence of SEQ ID NO: 3 described above. Shown to include.

Recombinant antibodies according to embodiments can target both PD-1 and CD47, thereby significantly enhancing stimulation of the immune system and providing stronger tumor suppressor capacity than antibodies to a single target. Can be shown.

In a third aspect, the nucleic acid is provided in the present disclosure. In embodiments of the present disclosure, the nucleic acid encodes the recombinant antibody, as described above. The recombinant antibody encoded by the nucleic acid according to the embodiment can target both PD-1 and CD47, thereby significantly enhancing stimulation of the immune system and more than an antibody against a single target. Can also show strong tumor suppressive ability.

In embodiments of the present disclosure, the nucleic acids described above may further comprise at least one of the following additional technical features:

In embodiments of the present disclosure, the nucleic acid comprises the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 5.

According to the first aspect, the second aspect, and the disclosure in this embodiment, the nucleotide sequence of SEQ ID NO: 4 is a light of the anti-PD-1 antibody of SEQ ID NO: 2 described in the second aspect. It is shown to encode a chain.

According to the first aspect, the second aspect, and the disclosure in the present embodiment, the nucleotide sequence of SEQ ID NO: 5 comprises the heavy chain of the recombinant antibody of SEQ ID NO: 3 described in the second aspect. Shown to code.

According to the present embodiment, the heavy chain of the anti-PD-1 antibody containing the amino acid sequence of SEQ ID NO: 6 described in the second aspect is contained in the nucleotide sequence of SEQ ID NO: 5 described above. It is shown to be encoded by the nucleotide sequence of number 8.

According to the present embodiment, the linker containing the amino acid sequence of SEQ ID NO: 1 described in the first aspect is encoded by the nucleotide sequence of SEQ ID NO: 9 contained in the nucleotide sequence of SEQ ID NO: 5 described above. It is shown that it has been done.

According to the present embodiment, the human SIRPA extracellular segment containing the amino acid sequence of SEQ ID NO: 7 described in the second aspect is contained in the nucleotide sequence of SEQ ID NO: 5 described above of SEQ ID NO: 10. It is shown to be encoded by the nucleotide sequence.

Recombinant antibodies encoded by nucleic acids according to embodiments can target both PD-1 and CD47, thereby significantly enhancing stimulation of the immune system and more than antibodies to a single target. It can show strong tumor suppressive ability.

In a fourth aspect, the construct is provided in the present disclosure. In embodiments of the present disclosure, the construct comprises a first nucleic acid molecule encoding the anti-PD-1 antibody and a second nucleic acid molecule encoding the human SIRPA extracellular segment. After the construct in the embodiment is introduced into the recipient cell, the recombinant antibody expressed can target both PD-1 and CD47, thereby significantly enhancing stimulation of the immune system. It can exhibit stronger tumor suppressor ability than antibodies against a single target.

In embodiments of the present disclosure, the construct described above may further include at least one of the following additional technical features:

In embodiments of the present disclosure, the construct further comprises a first promoter operably linked to said first nucleic acid molecule. The first promoter can effectively initiate the expression of the first nucleic acid molecule and the second nucleic acid molecule, thereby achieving the expression of the recombinant antibody.

In embodiments of the present disclosure, the construct described above may further include at least one of the following additional technical features:

In embodiments of the present disclosure, the first promoter is selected from U6, H1, CMV, EF-1, LTR, or RSV promoters. The present inventors can effectively initiate the expression of the first nucleic acid molecule and the second nucleic acid molecule by the U6, H1, CMV, EF-1, LTR, or RSV promoter. The expression efficiency of the nucleic acid molecule 1 and the nucleic acid molecule 2 is also significantly improved.

In embodiments of the present disclosure, the construct further comprises a third nucleic acid molecule, wherein the third nucleic acid molecule is located between the first nucleic acid molecule and the second nucleic acid molecule. It is configured to code the linker. Therefore, the first protein expressed by the first nucleic acid molecule and the second protein expressed by the second nucleic acid molecule can be bound together via the linker, and the fusion protein. It functions as and shows its effectiveness.

In embodiments of the present disclosure, the linker comprises the amino acid sequence of SEQ ID NO: 1.

In the embodiments of the present disclosure, the vector of the construct is a non-pathogenic viral vector. Since the pathogenic site of the construct vector in this embodiment has been modified or mutated, the vector is a non-pathogenic virus. Therefore, the treatment via the non-pathogenic viral vector according to the present embodiment is highly safe.

In embodiments of the present disclosure, the viral vector comprises at least one selected from retroviral vectors, lentiviral vectors, or adenovirus-related viral vectors. The vectors described above can achieve highly efficient expression of the transported nucleic acid in recipient cells with high processing efficiency.

In a fifth aspect, the method of preparing the recombinant antibody described above is provided in the present disclosure. In the embodiments of the present disclosure, the method comprises introducing the construct into a mammalian cell and culturing the mammalian cell under conditions suitable for protein expression and secretion to obtain the recombinant antibody. including. The method according to the embodiment can easily and efficiently obtain the recombinant antibody described above. As described above, the recombinant antibody can target both PD-1 and CD47, thereby significantly enhancing stimulation of the immune system and more than antibodies to a single target. It can show strong tumor suppressive ability.

In embodiments of the present disclosure, the methods described above may further include at least one of the following additional technical features:

In the embodiments of the present disclosure, the mammalian cell comprises at least one selected from HEK1, CHOS, 293F, and 293T.

In a sixth aspect, a therapeutic composition for treating cancer is provided in the present disclosure. In embodiments of the present disclosure, the therapeutic composition comprises the construct, recombinant antibody, or nucleic acid described above. The therapeutic composition according to the embodiment exhibits a significantly increased therapeutic effect in cancer.

In embodiments of the present disclosure, the therapeutic composition administered to the patient is preferably applied in a biocompatible solution or a pharmaceutically acceptable carrier. The various therapeutic compositions prepared may be suspended or dissolved in a pharmaceutically or physiologically acceptable carrier such as saline, isotonic saline, or other formulations apparent to those of skill in the art. can. Suitable carriers are largely dependent on the route of administration. Other aqueous or non-aqueous isotonic sterilized injections and aqueous or non-aqueous sterilized suspensions are pharmaceutically acceptable carriers.

In a seventh aspect, a method of treating cancer is provided in the present disclosure. In embodiments of the present disclosure, the method comprises administering to a patient suffering from cancer the construct, recombinant antibody, or nucleic acid described above. The methods according to the embodiments of the present disclosure exhibit a significantly increased therapeutic effect in cancer.

Based on the above, one object of the present disclosure is to provide novel bispecific antibodies that can target both PD1 and CD47.

CD47, also known as an integrin-related protein (IAP), is a transmembrane glycoprotein with a molecular weight of approximately 50 kDa and belongs to the immunoglobulin (Ig) superfamily. CD47 contains an N-terminal extracellular IgV domain, five highly hydrophobic transmembrane segments, and a C-terminal cytoplasmic region, and interacts with signal regulatory proteins α (SIRPα), thrombospondin-1 (TSP1), and integrins. It can act and is involved in the phagocytosis and control of transmembrane migration of immune cells such as T cells and monospheres.

The CD47 ligand, also known as the CD172a or SH2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), the SIRPα protein, is a transmembrane glycoprotein belonging to the immunoglobulin superfamily and is the N-terminus of the SIRPα protein. Is bound to CD47. The SIRPα protein is widely expressed in dendritic cells (DCs), macrophages, mast cells, granulocytes, nerve cells, and hematopoietic stem cells, but less in other cells. Binding of SIRPα protein to CD47 results in phosphorylation of the immunoreceptor tyrosine inhibitory motif (ITIM) in the C-terminal cytoplasmic region, resulting in mobilization and phosphorylation of tyrosine phosphatases SHP-1 and SHP-2, downstream signaling. It activates transduction pathways, thereby inhibiting macrophage feeding.

CD47 is widely expressed on the surface of various types of cells and emits a "don't eat me" signal. CD47 is an important self-labeling protein on the cell surface that emits important signals to regulate the phagocytosis of macrophages. CD47 can bind to the SIRPα protein on the surface of macrophages, trigger phosphorylation of ITIM and then recruitment of the SHP-1 protein, producing a series of cascade reactions, thereby inhibiting phagocytosis of macrophages. CD47 is highly expressed in active erythrocytes and low in aged erythrocytes, allowing specific attacks by macrophages and clearance of aged erythrocytes. It has been found that tumor cells can use CD47s (which emit "don't eat me" signals) to escape the attack of the body's immune system. Another study showed that CD47 was first identified as a tumor antigen for human ovarian cancer in the 1980s, after which CD47 has significantly higher expression levels than normal cells, acute myelogenous leukemia (AML). Expressed in a variety of human tumor types, including chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myelogenous tumors (MM), and other solid tumors. It was also found that. Therefore, the CD47 signaling pathway and its ligand SIRPα protein have become new targets for inhibiting immune escape in tumor cells. In recent years, drug development based on this signal transduction pathway has been attracting attention.

There are various mechanisms that block the binding of CD47 to the SIRPα protein via drugs that suppress tumor immune escape. First, physical blockade of CD47 binding to SIRPα protein can alleviate inhibition of SIRPα protein on macrophages independent of cytotoxic effects mediated by Fc fragments of antibodies belonging to non-innate immunity. can. It was discovered that anti-CD47 antibodies lacking the Fc fragment could still promote tumor clearance by macrophages. Second, blocking CD47 can directly induce tumor apoptosis without depending on macrophages. Third, the anti-CD47 antibody can initiate anti-tumor adaptive immunity by inducing antigen presentation and activating both SIRPα-expressing T cells and DC cells. Blocking the signaling pathway between CD47 and SIRPα can alleviate the inhibition of cytokine production and DC maturation. DC cells can kill tumor cells through the synergistic effect of CD47 antibody and phagocytic cells, presenting tumor-related antigens to CD8 + T cells, thereby exerting a specific killing effect on CD8 + T cells on the tumor.

Therefore, the CD47 / SIRPα antibody is an important next-generation tumor immunosuppressive drug product after the PD1 / PD-L1 antibody. CD47 and PD-L1 have similarities, for example, both of which are regulated by the transcription factor myc and are widely expressed in various tumor cells. In some embodiments, the CD47 antibody may be more promising than the anti-PD1 / PD-L1 antibody. Specifically, CD47 is more widely expressed than PD-L1 and is highly expressed in almost all tumor cells, indicating a broader range of efficacy. Furthermore, the CD47 antibody exerts tumor suppression through a variety of mechanisms over the PD-1 antibody. For example, the fact that antibodies against conventional immunosuppressive agents such as PD-1 and CTLA-4 do not kill T cells in a tumor-specific manner has a limited function such that it has an effect on only a small part of tissue. CD47 antibody, on the other hand, can initiate macrophage-mediated non-adaptive immunity, as well as specific killing of tumor cells through antigen presentation of macrophages, DC cells, etc. You can also start. However, due to its widespread expression, CD47 is also associated with the regulation of non-immune signals in various tissues, thereby blocking the CD47 signal, causing macrophages to extensively attack their normal tissues or impaired non-immune regulation. May bring. Phagocytosis by macrophages requires a synergistic effect of a "don't eat me" signal expressed by CD47 or the like and a "eat me" signal expressed by calreticulin (CRT) or the like. In general, tumor cells highly express CRT, but normal cells do not express CRT and "eat me" signals, so CD47 is widely present in the human cerebral cortex, cerebellum, and other healthy tissues. However, the CD47 antibody shows only a limited blocking effect in normal tissues. However, according to clinical data from Phase I clinical trials, patients receiving radiation and chemotherapy express an up-regulated "eat me" signal. After CD47 antibody treatment, CD47 + red blood cells are depleted, which may cause transient anemia as the main side effect.

CD47 antibody therapy exerts a tumor-killing effect via DC cells and CD8 + T cells. DC cells can kill tumor cells through the synergistic effect of CD47 antibody and phagocytic cells, presenting tumor-related antigens to CD8 + T cells, thereby exerting a specific killing effect of CD8 + T cells on the tumor. Tumor cells upregulate the expression of CD47 to deceive macrophages, whereas CD47 antibodies can block the "don't eat me" signal expressed by CD47 to exert phagocytosis by macrophages. can. Recombinant antibodies of the present disclosure can target both PD-1 and CD47, thereby significantly enhancing stimulation of the immune system and exhibiting stronger tumor suppressor capacity, the next generation. Serves as an immune checkpoint inhibitor.

Further aspects and advantages of the present disclosure will be described in part in the description below, some of which will be apparent from the description below or will be understood from the practice of the present disclosure.

FIG. 1 is a result showing the identification of HX009-5 by SDS-PAGE according to the embodiment of the present disclosure. FIG. 2 is a result showing the identification of HX009-5 by SEC-HPLC according to the embodiment of the present disclosure. FIG. 3 is a diagram showing experimental results regarding the affinity of H8 and HX009-5 for PD-1 according to the embodiment of the present disclosure. FIG. 4 is a diagram showing the results of inhibition of binding between PD-1 and PD-L1 by HX009-5 or H8 according to the embodiment of the present disclosure. FIG. 5 is a diagram showing the results of inhibition of binding between PD-1 and PD-L2 by HX009-5 or H8 according to the embodiment of the present disclosure. FIG. 6 is a diagram showing the experimental results regarding the binding between HX009-5 and hCD47 according to the embodiment of the present disclosure. FIG. 7 is a diagram showing the experimental results regarding the suppression of the binding between CD47 and SIRPA by HX009-5 according to the embodiment of the present disclosure. FIG. 8 is a diagram showing the experimental results when there is no bond between HX009-5 and CD16a according to the embodiment of the present disclosure. FIG. 9 is a diagram showing the experimental results in the case where there is no bond between HX009-5 and CD32a according to the embodiment of the present disclosure. FIG. 10 is a diagram showing the experimental results when there is no bond between HX009-5 and CD32b according to the embodiment of the present disclosure. FIG. 11 is a diagram showing the experimental results in the case where there is no bond between HX009-5 and CD64 according to the embodiment of the present disclosure. FIG. 12 is a diagram showing the experimental results relating to the stimulation of T lymphocytes for the secretion of IL-2 by HX009-5 or H8 according to the embodiment of the present disclosure. FIG. 13 is a diagram showing the experimental results regarding the absence of the hemagglutination reaction generated by HX009-5 according to the embodiment of the present disclosure. FIG. 14 is a diagram showing the results regarding the change in tumor volume over time according to the embodiment of the present disclosure. FIG. 15 is a diagram showing the results regarding changes in tumor volume over time according to the embodiment of the present disclosure.

Examples of the present disclosure are described in detail below.

The embodiments of the present disclosure will be referred to in detail. Those skilled in the art will appreciate that the following examples are for illustration purposes only and cannot be construed as limiting the scope of this disclosure. Unless specific techniques or conditions are specified in the examples, see literature in the art (eg, J. Sambrook et al. (Translated by Hung PT), Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press). ), Or according to the product description. Unless the manufacturer of the reagent or instrument is specified, the reagent or instrument may be commercially available, for example, from the Illumina Company.

The cell lines and general experimental techniques used in the following examples are as follows.

Example 1 Protein expression of PD-1 / CD47 bispecific antibody Based on humanized antibody H8 (anti-PD-1 IgG antibody), via a specific linker (GGGGSGGGGSERGETGP), human signal regulatory protein alpha (SIRPA, NP_542970) .1) By binding the extramembrane domain (32aa-137aa) to the C-terminal of the H8 heavy chain, a bispecific antibody HX009-5 is obtained, whereby the bispecific antibody is the protein of interest. Target both PD-1 and CD47.

In a practical procedure, the entire sequence of nucleic acid encoding the light chain of humanized antibody H8 is synthesized and then inserted into the vector to obtain expression vector 1; the entire sequence of nucleic acid encoding the H8 heavy chain and The entire sequence of the nucleic acid encoding the linker-SIRPA ( 118 mer) was also synthesized, and the nucleic acid encoding the H8 heavy chain was directly inserted into the expression vector 1 to obtain an expression vector 2 expressing the anti-PD1 monoclonal antibody H8. rice field. After fusion by overlap PCR, a nucleic acid encoding a fusion peptide of the heavy chain of anti-PD1 monoclonal antibody H8 and linker-SIRPA is inserted into expression vector 1, thereby expressing bispecific antibody HX009-5. I got 3. Then, the DNA extracted from the expression vector 2 and the expression vector 3 was transfected into mammalian cells (293 cells), respectively. By such transfection, the antibody was expressed in mammalian cells and secreted from the cells. Then, after purification on an antibody A affinity chromatography column, proteins H8 and HX009-5 were obtained. HX009-5 was used for subsequent pharmacodynamic studies after quality identification by SDS-PAGE and SEC-HPLC standard analysis techniques.

1 and 2 are the results showing the identification of HX009-5 by SDS-PAGE and SEC-HPLC, respectively.

FIG. 1 shows the SDS-PAGE results for the identification of HX009-5. In FIG. 1, lane 1 represents HX009-5 (reduced); lane 2 represents H8 (reduced); lane M represents a protein marker (18.4KDa; 25KDa; 35KDa; 45KDa; Represents 66.2KDa); Lane 3 represents a bovine serum albumin (BSA) buffer. As shown in FIG. 1, the candidate sample for antibody HX009-5 exhibits a relatively high overall purity.

FIG. 2 shows the SEC-HPLC results in the identification of HX009-5. As shown in FIG. 2, it has been confirmed by integral quantification that the total purity of the antibody is 98.2%.

可変領域に下線が引かれる。 HX009-5 heavy chain amino acid sequence

The variable area is underlined.

Nucleic acid sequence encoding heavy chain of HX009-5

可変領域に下線が引かれる。 Amino acid sequence of light chain of HX009-5

The variable area is underlined.

Nucleic acid sequence encoding the light chain of HX009-5

Example 2 Elisa binding assay for HX009-5 bispecific antibody 1. PD-1 Binding ELISA Assay for H8 and HX009-5 A direct comparison was made between the H8 and HX009-5 antibodies obtained in Example 1, including the PD-1 binding assay and PD-L1 competing assay. rice field. The details are as follows.

The specific steps for the PD-1 binding assay are as follows.
1) Antibody coating ELISA plates were coated with hPD-1-his antigen at a concentration of 0.25 μg / ml (100 μl per well) and incubated overnight at 4 ° C.
2) Blocking Using 1% BSA (diluted in PBS buffer), an ELISA plate coated with hPD-1-his antigen was blocked at 37 ° C. for 2 hours, and then 3 in 1 × PBST buffer containing 1% Tween-20. Washed twice and tapped gently to dry.
3) Incubation with primary antibody H8 antibody and HX009-5 antibody were serially diluted 1: 5 from 2 μg / ml, respectively, to obtain 7 gradient antibody solutions for each antibody. Seven gradient antibody solutions for each antibody and a blank PBS control were added to each blocked ELISA plate and incubated at 37 ° C. for 1 hour.
4) Incubation with secondary antibody The ELISA plate was washed 3 times with PBST buffer, gently tapped to dry, and then as secondary antibody, 1: 10000 diluted goat anti-human IgG-HRP (H + L) (per well). 100 μl) was added and incubated at 37 ° C. for 1 hour.
5) Coloring (developing)
The ELISA plate was washed 3 times with PBST buffer, tapped gently again to dry, and then 100 μl of 3,3', 5,5'-tetramethylbenzidine (TMB) as a developer was added per well. Incubated at room temperature for 5 to 15 minutes.
6) Termination of color development The color development was terminated by adding _{50 μl of 2M H 2} SO _{4 solution per well.}
7) Reading
On a microplate reader, the absorbance of the solution in each well was measured under a wavelength of 450 nm.

As shown in Table 1 and Figure 3, _{EC 50} values the H8 and HX009-5 that bind to PD-1 can be calculated as both are 0.05 nM. FIG. 3 shows that fusion of H8 with a linker-SIRPA ( 118 mer) at the C-terminus does not result in changes in antibody HX009-5 binding affinity for PD-1.

2. PD-L1 Competitive ELISA Assay for H8 and HX009-5 Specific steps are as follows.
1) Antibody coating A 96-well ELISA plate was coated with hPD-1-hIgGFc antigen at a concentration of 0.5 μg / ml (50 μl per well) and incubated overnight at 4 ° C.
2) Blocking After washing 3 times with PBST buffer and tapping gently to dry, the 96-well ELISA plate coated with 1% BSA (diluted with PBS buffer) was blocked at 37 ° C. for 2 hours, and then 1%. It was washed 3 times with 1 × PBST buffer containing Tween-20.
3) Incubation with primary antibody H8 antibody and HX009-5 antibody were serially diluted 1: 3 from 6 μg / ml, respectively, to obtain 7 gradient antibody solutions for each antibody. Seven gradient antibody solutions for each antibody and blank PBS control (50 μl per well) were added to each blocked 96-well ELISA plate and incubated for 10 minutes at room temperature.
4) Incubation with ligand To 50 μl per well, 0.6 μg / ml PD-L1-mIgG2aFc solution was added, and the mixture was incubated at 37 ° C. for 1 hour.
5) Incubation with secondary antibody A 96-well ELISA plate was washed 3 times with PBST buffer, gently tapped to dry, and then 1: 5000 diluted goat anti-mouse IgG-HRP (H + L) as secondary antibody (H + L). 50 μl per well) was added and incubated at 37 ° C. for 1 hour.
6) Color development A 96-well ELISA plate was washed 3 times with PBST buffer, tapped gently again to dry, and then 50 μl of TMB as a color-developing agent was added per well and incubated for 5 to 15 minutes at room temperature.
7) Termination of color development The color development was terminated by adding _{50 μl of 2M H 2} SO _{4 solution per well.}
8) Reading On a microplate reader, the absorbance of the solution in each well was measured under a wavelength of 450 nm.

The results are shown in Table 2 and FIG. HX009-5 antibody and H8 antibody for inhibition of PD-1 binding to PD-L1, it indicates the respective _{IC 50} values of 1.5nM and 1.0 nM, linker -SIRPA at the C-terminus of H8 (118 mer) Fusion with antibody HX009-5 does not result in a clear change in the inhibition of PD-1 binding to PD-L1.

3. 3. PD-L2 Competitive ELISA Assay for H8 and HX009-5 Specific steps are as follows.
1) Antibody coating A 96-well ELISA plate was coated with a concentration of 1.0 μg / ml (100 μl per well) of hPD-1-hIgGFc antigen and incubated overnight at 4 ° C.
2) Blocking After washing 3 times with PBST buffer and tapping gently to dry, the 96-well ELISA plate coated with 1% BSA (diluted with PBS buffer) was blocked at 37 ° C. for 2 hours, and then 1%. It was washed 4 times with 1 × PBST buffer containing Tween-20.
3) Incubation with primary antibody H8 antibody and HX009-5 antibody were serially diluted 1: 3 from 20 μg / ml, respectively, to obtain 7 gradient antibody solutions for each antibody. Seven gradient antibody solutions for each antibody and blank PBS control (50 μl per well) were added to each blocked 96-well ELISA plate and incubated for 10 minutes at room temperature.
4) Incubation with ligand 50 μl of PD-L2-his tag solution of 0.6 μg / ml was added per well, and the mixture was incubated at 37 ° C. for 1 hour.
5) Incubation with secondary antibody The 96-well ELISA plate was washed 5 times with PBST buffer, gently tapped to dry, and then diluted 1: 750 (50 μl per well) to anti-HRP binding as the secondary antibody. A his tag mouse monoclonal antibody was added and incubated at 37 ° C. for 1 hour.
6) Color development A 96-well ELISA plate was washed 6 times with PBST buffer, tapped gently again to dry, and then 100 μl of TMB as a color former was added per well and incubated for 30 minutes at room temperature.
7) Termination of color development The color development was terminated by adding _{50 μl of 2M H 2} SO _{4 solution per well.}
8) Reading On a microplate reader, the absorbance of the solution in each well was measured under a wavelength of 450 nm.

The results are shown in Table 3 and FIG. _{The HX009-5 and H8 antibodies showed IC 50} values of 1.5 nM and 2.7 nM, respectively, for inhibition of PD-1 binding to PD-L2, and linker-SIRPA (118 mer) at the C-terminus of H8. Fusion with antibody HX009-5 does not result in a clear change in the inhibition of PD-1 binding to PD-L2.

4. CD47-Binding ELISA Assay for HX009-5 A CD47-Binding ELISA assay for the HX009-5 antibody obtained in Example 1 was performed. Details are given below.

The specific process is as follows.
1) Antibody-coated ELISA plate was coated with CD47 antigen at a concentration of 0.25 μg / ml (100 μl per well) and incubated overnight at 4 ° C.
2) Blocking The ELISA plate coated with CD47 antigen was blocked with 1% BSA (diluted with PBS buffer) for 2 hours at 37 ° C., and then washed 3 times with 1 × PBST buffer containing 1% Tween-20. Gently tap to dry.
3) Incubation with primary antibody The HX009-5 antibody was serially diluted 1: 5 from 10 μg / ml to obtain 7 gradient antibody solutions. Seven gradient antibody solutions and a blank PBS control were added to each blocked ELISA plate and incubated at 37 ° C. for 1 hour.
4) Incubation with secondary antibody The ELISA plate was washed 3 times with PBST buffer, gently tapped to dry, and then as secondary antibody, 1: 10000 diluted goat anti-human IgG-HRP (H + L) (per well). 100 μl) was added and incubated at 37 ° C. for 1 hour.
5) Color development The ELISA plate was washed 3 times with PBST buffer, tapped gently again to dry, and then 100 μl of TMB as a color developing agent was added per well and incubated for 5 to 15 minutes at room temperature.
6) Termination of color development The color development was terminated by adding _{50 μl of 2M H 2} SO _{4 solution per well.}
7) Reading On a microplate reader, the absorbance of the solution in each well was measured under a wavelength of 450 nm.

The results are shown in Table 4 and FIG. HX009-5 and _{EC 50} in the binding between hCD47 is 0.6 nM.

5. SIPRA Competitive ELISA Assay for HX009-5 The specific steps are as follows.
1) Antibody coating A 96-well ELISA plate was coated with a CD47 antigen at a concentration of 0.25 μg / ml (100 μl per well) and incubated overnight at 4 ° C.
2) Blocking After washing 3 times with PBST buffer and tapping gently to dry, block the applied 96-well ELISA plate with 1% BSA (diluted with PBS buffer) at 37 ° C. for 2 hours and 1% Tween. It was washed 4 times with 1 × PBST buffer containing -20.
3) Incubation with primary antibody The HX009-5 antibody was serially diluted 1: 3 from 30 μg / ml to obtain 7 gradient antibody solutions. Seven gradient antibody solutions and blank PBS control (50 μl per well) were added to each blocked 96-well ELISA plate and incubated for 10 minutes at room temperature.
4) Incubation with ligand 50 μl of a 0.6 μg / ml SIRPA-his tag solution was added per well, and the mixture was incubated at 37 ° C. for 1 hour.
5) Incubation with secondary antibody The 96-well ELISA plate was washed 5 times with PBST buffer, gently tapped to dry, and then diluted 1: 750 (50 μl per well) to anti-HRP binding as the secondary antibody. A his tag mouse monoclonal antibody was added and incubated at 37 ° C. for 1 hour.
6) Color development A 96-well ELISA plate was washed 6 times with PBST buffer, tapped gently again to dry, and then 100 μl of TMB as a color former was added per well and incubated for 30 minutes at room temperature.
7) Termination of color development The color development was terminated by adding _{50 μl of 2M H 2} SO _{4 solution per well.}
8) Reading On a microplate reader, the absorbance of the solution in each well was measured under a wavelength of 450 nm.

The results shown in Tables 5 and 7, HX009-5 is an _{IC 50} of 21 nM, indicating that inhibits binding between CD47 and Sirpa.

Example 3 Study on the efficacy of the HX009-5 Fc fragment To investigate the efficacy of the Fc fragment of HX009-5 (obtained in Example 1), determine the binding capacity of HX009-5 to individual FC receptors. The affinity of each Fc receptor (CD16, CD32a, CD32b, and CD64) was tested. Details are given below.

1. 1. Assay for HX009-5 Affinity for CD16a The Fc receptor CD16a (also known as FcγRIIIa) can bind to the Fc fragment of an IgG antibody and participate in antibody-dependent cellular cytotoxicity (ADCC). The ability of the therapeutic monoclonal antibody to bind to the Fc receptor affects the safety and efficacy of the antibody. In this example, the affinity of HX009-5 for CD16a was tested by ELISA and the ability of HX009-5 to bind to the Fc receptor CD16a was evaluated.

The HX009-5 antibody (obtained in Example 1) was detected by ELISA binding to CD16a as compared to the HX006 antibody (IgG1 subtype). Details are given below.

The specific process is as follows.
1) Antibody-coated ELISA plate was coated with CD16a antigen at a concentration of 0.5 μg / ml (100 μl per well) and incubated overnight at 4 ° C.
2) Blocking The ELISA plate coated with the CD16a antigen was blocked with 1% BSA (diluted with PBS buffer) at 37 ° C. for 2 hours, and then washed 3 times with 1 × PBST buffer containing 1% Tween-20. Gently tap to dry.
3) Incubation with primary antibody The HX009-5 antibody was serially diluted 1: 5 from 10 μg / ml to obtain 7 gradient antibody solutions. Seven gradient antibody solutions and a blank PBS control were added to each blocked ELISA plate and incubated at 37 ° C. for 1 hour.
4) Incubation with secondary antibody The ELISA plate was washed 3 times with PBST buffer, gently tapped to dry, and then as secondary antibody, 1: 8000 diluted goat anti-human IgG-HRP (H + L) (per well). 100 μl) was added and incubated at 37 ° C. for 1 hour.
5) Color development The ELISA plate was washed 3 times with PBST buffer, tapped gently again to dry, and then 100 μl of TMB as a color developing agent was added per well and incubated for 30 minutes at room temperature.
6) Termination of color development The color development was terminated by adding _{50 μl of 2M H 2} SO _{4 solution per well.}
7) Reading On a microplate reader, the absorbance of the solution in each well was measured under a wavelength of 450 nm.

The results shown in Table 6 and FIG. 8 show that there is no significant binding between HX009-5 and CD16a.

2. Assay for HX009-5 Affinity for CD32a The HX009-5 antibody (obtained in Example 1) was compared to the HX006 antibody (IgG1 subtype) and binding to CD32a was detected by ELISA. Details are given below.

The specific process is as follows.
1) Antibody-coated ELISA plate was coated with CD32a antigen at a concentration of 0.5 μg / ml (100 μl per well) and incubated overnight at 4 ° C.
2) Blocking An ELISA plate coated with CD32a antigen is blocked with 1% BSA (diluted with PBS buffer) at 37 ° C. for 2 hours, washed 3 times with 1 × PBST buffer containing 1% Tween-20, and gently washed. It was beaten to dry.
3) Incubation with primary antibody The HX009-5 antibody was serially diluted 1: 5 from 10 μg / ml to obtain 7 gradient antibody solutions. Seven gradient antibody solutions and a blank PBS control were added to each blocked ELISA plate and incubated at 37 ° C. for 1 hour.
4) Incubation with secondary antibody Goat anti-human IgG-HRP as secondary antibody at 1: 8000 dilution (100 μl per well) after washing the ELISA plate 3 times with PBST buffer and tapping gently to dry. (H + L) was added and incubated at 37 ° C. for 1 hour.
5) Color development The ELISA plate was washed 3 times with PBST buffer, tapped gently again to dry, and then 100 μl of TMB as a color developing agent was added per well and incubated for 30 minutes at room temperature.
6) Termination of color development The color development was terminated by adding _{50 μl of 2M H 2} SO _{4 solution per well.}
7) Reading On a microplate reader, the absorbance of the solution in each well was measured under a wavelength of 450 nm.

The results shown in Table 7 and FIG. 9 show that there is no significant binding between HX009-5 and CD32a.

3. 3. Assay for HX009-5 Affinity for CD32b The HX009-5 antibody (obtained in Example 1) was compared to the HX006 antibody (IgG1 subtype) and binding to CD32b was detected by ELISA. Details are given below.

The specific process is as follows.
1) Antibody-coated ELISA plate was coated with CD32b antigen at a concentration of 0.5 μg / ml (100 μl per well) and incubated overnight at 4 ° C.
2) Blocking An ELISA plate coated with CD32b antigen is blocked with 1% BSA (diluted with PBS buffer) at 37 ° C. for 2 hours, then washed 3 times with 1 × PBST buffer containing 1% Tween-20 and gently. It was beaten to dry.
3) Incubation with primary antibody The HX009-5 antibody was serially diluted 1: 5 from 10 μg / ml to obtain 7 gradient antibody solutions. Seven gradient antibody solutions and a blank PBS control were added to each blocked ELISA plate and incubated at 37 ° C. for 1 hour.
4) Incubation with secondary antibody Goat anti-human IgG-HRP as secondary antibody at 1: 8000 dilution (100 μl per well) after washing the ELISA plate 3 times with PBST buffer and tapping gently to dry. (H + L) was added and incubated at 37 ° C. for 1 hour.
5) Color development The ELISA plate was washed 3 times with PBST buffer, tapped gently again to dry, and then 100 μl of TMB as a color developing agent was added per well and incubated for 30 minutes at room temperature.
6) Termination of color development The color development was terminated by adding _{50 μl of 2M H 2} SO _{4 solution per well.}
7) Reading On a microplate reader, the absorbance of the solution in each well was measured under a wavelength of 450 nm.

The results shown in Table 8 and FIG. 10 show that there is no significant binding between HX009-5 and CD32b.

4. Assay for HX009-5 Affinity for CD64 The HX009-5 antibody (obtained in Example 1) was compared to the HX006 antibody (IgG1 subtype) and binding to CD64 was detected by ELISA. Details are given below.

The specific process is as follows.
1) Antibody-coated ELISA plate was coated with a CD64 antigen at a concentration of 0.5 μg / ml (100 μl per well) and incubated overnight at 4 ° C.
2) Blocking An ELISA plate coated with CD64 antigen is blocked with 1% BSA (diluted with PBS buffer) at 37 ° C. for 2 hours, then washed 3 times with 1 × PBST buffer containing 1% Tween-20 and gently. It was beaten to dry.
3) Incubation with primary antibody The HX009-5 antibody was serially diluted 1: 5 from 10 μg / ml to obtain 7 gradient antibody solutions. Seven gradient antibody solutions and a blank PBS control were added to each blocked ELISA plate and incubated at 37 ° C. for 1 hour.
4) Incubation with secondary antibody Goat anti-human IgG-HRP as secondary antibody at 1: 8000 dilution (100 μl per well) after washing the ELISA plate 3 times with PBST buffer and tapping gently to dry. (H + L) was added and incubated at 37 ° C. for 1 hour.
5) Color development The ELISA plate was washed 3 times with PBST buffer, tapped gently again to dry, and then 100 μl of TMB as a color developing agent was added per well and incubated for 30 minutes at room temperature.
6) Termination of color development The color development was terminated by adding _{50 μl of 2M H 2} SO _{4 solution per well.}
7) Reading On a microplate reader, the absorbance of the solution in each well was measured under a wavelength of 450 nm.

The results shown in Table 9 and FIG. 11 show that there is no significant binding between HX009-5 and CD64.

Example 4 Biological activity of bispecific antibody and anti-PD1 antibody by mixed lymphocyte reaction (MLR) Stimulates T lymphocytes by mixed lymphocyte reaction (MLR) to secrete IL-2 and IFN-γ. The ability to do so was detected between HX009-5 (obtained in Example 1) and H8. Details are shown below.

In mixed lymphocyte reaction, after mixing with dendritic cells (DC) from another individual, T cells (TC) were stimulated and IL-2 and IFN-γ were secreted by DC presentation. First, monocytes in the blood were induced to differentiate into DC by the cytokines GM-CSF and IL-4. The DC was then stimulated by TNFa to mature. Secretory levels of IL-2 and IFN-γ in cell culture supernatants were detected 5 days after mixing mature DC and heterologous TC. Specifically, TC at a concentration of 1 × 10 ^{5 cells / well and DC at a concentration of 1 × 10 4} cells / well were mixed in a 96-well plate and antibodies with eight gradient concentrations from 10M to 0.09765625nM. Was added. After 5 days, the IL-2 detection kit was used to quantify the IL-2 content of the supernatant.

FIG. 12 shows the level of IL-2 secretion in T lymphocytes stimulated by H8 antibody and HX009-5 antibody, respectively. As can be seen from FIG. 12, the H8 and HX009-5 antibodies are capable of effectively stimulating T lymphocytes to secrete IL-2, with the linker-SIRPA ( 118 mer) at the C-terminal of H8. It is shown that the fusion of T lymphocytes does not bring about a change in antibody HX009-5 in stimulating T lymphocytes to secrete IL-2.

Example 5 Study on hemagglutination reaction by HX009-5 The specific steps are as follows.
1. 1. From volunteers, 5 ml of peripheral blood was collected in an anticoagulant-treated tube with 5 ml of heparin and gently shaken for good contact. The blood was transferred to a 15 ml centrifuge tube, mixed with 9 ml PBS under gentle shaking, and then the peripheral blood was centrifuged at 2100 rpm for 10 minutes.
2. The leukocyte-containing supernatant above the erythrocyte layer was discarded, 12 ml PBS was added for resuspension, and then centrifuged at 1500 rpm for 5 minutes.
3. 3. The supernatant above the erythrocyte layer was discarded, 12 ml PBS was added for resuspension, and the mixture was centrifuged at 1500 rpm for 5 minutes.
4. Step 3 was repeated twice.
5. After discarding the supernatant, 1 ml erythrocyte suspension was transferred to a 15 ml centrifuge tube and mixed with 9 ml PBS to prepare for use as 10% erythrocytes in PBS.
6. 1 ml of 10% erythrocytes in PBS was mixed with 9 ml of PBS in a fresh 15 ml centrifuge tube to obtain 1% erythrocytes in PBS for use.
7. Preparation of Various Test Samples 1) HX009-5 was diluted from 9.1 mg / ml to 0.9 mg / ml and further serially diluted 1: 3 to obtain 12 gradient concentrations.
2) H8 was diluted from 10 mg / ml to 0.9 mg / ml and further serially diluted 1: 3 to obtain 12 gradient concentrations.
3) Potato lectin extract was serially diluted 1: 3 to give 8 gradient concentrations.
4) PBS was used as a blank control.

To each well of a 96-well U bottom plate B1 to G12, was added in a pattern indicated a test sample of 1% erythrocytes 50μl and 50 mu l of PBS in Table 10 below, 5% in an incubator of CO ₂ Incubated overnight at 37 ° C.

After 24 hours of incubation, 96-well plates were removed for observation with a gel imaging analyzer. As shown in Table 10 and FIG. 13, a clear hemagglutination reaction was observed in wells supplemented with the potato lectin extract solution (as a positive control) at the first four concentrations; while H8, HX009-5, Alternatively, no hemagglutination reaction was observed in wells to which PBS was added at various concentrations, indicating that the hemagglutination reaction was not triggered by HX009-5.

Example 6 Antitumor efficacy of HX009-5 in a Mixeno model subcutaneously transplanted with human anaplastic large cell lymphoma (KRAPAS-299) H8 and HX009 in a Mixeno model subcutaneously transplanted with human anaplastic large cell lymphoma (KRAPAS-299) For a study on antitumor efficacy of -5 (obtained in Example 1), a human tumor transplant model was established in NSG mice. Details are given below.

NSG mice characterized by deficiencies / mutations in NOD / Prkdcside / IL2rgnull are currently used as the most suitable mouse model of immunodeficiency for human cell transplantation, as there is little rejection of human cells and tissues. Therefore, we present the in vivo pharmacodynamics of HX009-5 in a graft-versus-host disease (GVHD) model established by adoptively introducing human peripheral blood mononuclear cells (PBMCs) into NSG mice. investigate. Using the NSG mouse-based human tumor transplantation model (Mixeno model), we investigate the antitumor efficacy of HX009-5 in the Mixeno model of subcutaneously transplanted human anaplastic large cell lymphoma (KRAPAS-299).

注：薬物投与量は、１０μｌ／ｇである。ｎは動物の数を表す。０日目は、腫瘍細胞が接種された日である。ｉ．ｖ．は、尾静脈を介した投与を表す。 On day 0, KARPAS-299 cells were subcutaneously inoculated into the right back of 30 NCG mice. On the 6th day after inoculation, when the tumor volume ^{reached an average value of 60 mm 3} , 30 NCG mice were divided into 5 groups, and 6 mice were assigned to each group. Human peripheral blood mononuclear cells (PBMC) from donor A are transplanted into 3 mice (marked "A") in each group via the tail vein; while humans from donor B Peripheral blood mononuclear cells (PBMCs) were transplanted into the remaining three mice in each group (marked as "B") via the tail vein. PBMCs from each donor were suspended in PBS for injection (injection volume: 0.1 ml / mouse). As shown in FIG. 11, 6 times on the 6th, 9th, 13th, 16th, 19th, and 22nd days after inoculation of the tumor cells, 5 groups via the tail vein. HX009-5 at individual doses of 0.1 mg / kg, 1 mg / kg, and 10 mg / kg to mice, and H8 at 10 mg / kg as a positive control (also referred to as HX008 in Table 11); and controls. Isoform antibody (human IgG4) at 5 mg / kg was administered. Treatment evaluation was performed by relative tumor suppression rate (TGI _RTV ), and safety evaluation was performed by changes in body weight and mortality.

Note: The drug dose is 10 μl / g. n represents the number of animals. Day 0 is the day when the tumor cells were inoculated. i. v. Represents administration via the tail vein.

14 and 15 show changes in tumor volume over time in the five groups. Administration of HX009-5 or H8 showed clear antitumor efficacy and dose dependence of HX009-5 was observed for the control group inoculated with PBMC and administered isoform antibody (human IgG4). rice field. That is, the higher the dose, the greater the tumor suppression. At the same dose, HX009-5 showed even stronger anti-tumor efficacy than H8, indicating that PD1 / CD47 dual target antibody is superior to PD1 single target antibody.

"Embodiments," "some embodiments," "one embodiment," "other examples," "examples," "concrete examples," or "some examples," etc. in the present specification. Reference means that a particular part, structure, material, or feature described in connection with an embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, "in some embodiments," "in one embodiment," "in embodiments," "in other embodiments," and "examples" described elsewhere throughout the specification. The appearance of expressions such as "in", "in specific examples", or "in some examples" does not necessarily mean the same as the embodiments or examples of the present disclosure. In addition, specific parts, structures, materials, or features may be combined in any suitable manner in one or more embodiments or examples.

Although descriptive embodiments have been shown and described, the above embodiments cannot be construed to limit the present disclosure, and the embodiments are modified, substituted, and within the scope of the present disclosure. It will be appreciated by those skilled in the art that modifications can be made.

Claims (18)

With anti-PD-1 antibody;
Human signal regulatory protein α (SIRPA) extracellular segment and
Recombinant protein containing
The N-terminus of the human SIRPA extracellular segment is connected to the C-terminus of the heavy chain of the anti-PD-1 antibody .
A recombinant protein, wherein the anti-PD-1 antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 2 and a heavy chain containing the amino acid sequence of SEQ ID NO: 6. The recombinant protein according to claim 1, wherein the anti-PD-1 antibody is an IgG-like antibody against PD-1. The recombinant protein further comprises a linker and contains
The set according to claim 1, wherein the N-terminus of the linker is connected to the C-terminus of the heavy chain of the anti-PD-1 antibody, and the C-terminus of the linker is connected to the N-terminus of the human SIRPA extracellular segment. Alternate protein. The recombinant protein according to claim 3, wherein the linker comprises the amino acid sequence of SEQ ID NO: 1. The recombinant protein according to claim 1, wherein the human SIRPA extracellular segment comprises the amino acid sequence of SEQ ID NO: 7. With a light chain containing the amino acid sequence of SEQ ID NO: 2;
A fusion peptide containing the amino acid sequence of SEQ ID NO: 3 and
Including
The recombinant protein according to claim 3, wherein the fusion peptide comprises a heavy chain of the anti-PD-1 antibody, the linker, and the human SIRPA extracellular segment. A nucleic acid comprising the recombinant protein according to any one of claims 1 to 6. The nucleic acid
With the first nucleotide sequence encoding the light chain of the anti-PD-1 antibody;
A second nucleotide sequence encoding the heavy chain of the anti-PD-1 antibody and the human SIRPA extracellular segment.
7. The nucleic acid according to claim 7. The nucleic acid according to claim 8, wherein the second nucleotide sequence further encodes a linker. The nucleic acid
With the first nucleotide sequence of SEQ ID NO: 4, which encodes the light chain of the anti-PD-1 antibody of SEQ ID NO: 2;
The second nucleotide sequence of SEQ ID NO: 5, which encodes the fusion peptide represented by SEQ ID NO: 3, and
Only including,
The nucleic acid according to claim 9 , wherein the fusion peptide comprises a heavy chain of the anti-PD-1 antibody, the linker, and the human SIRPA extracellular segment. A construct that expresses the recombinant protein according to any one of claims 1 to 6.
With the first nucleic acid molecule encoding the light chain of the anti-PD-1 antibody of claim 1;
It is seen containing a heavy chain of an anti-PD-1 antibody of claim 1, and a second nucleic acid molecule encoding a human SIRPA extracellular segment of claim 1, and
Optionally, the construct is further characterized in including that a promoter operably linked to said first nucleic acid molecule construct. The construct according to claim 11, wherein the second nucleic acid molecule further encodes a linker. The construct according to any one of claims 11 to 12, wherein the promoter is selected from U6, H1, CMV, EF-1, LTR, or RSV promoter. The vector of the construct is a non-pathogenic viral vector.
The construct according to any one of claims 11 to 13, wherein the viral vector comprises at least one selected from a retroviral vector, a lentiviral vector, or an adenovirus-related viral vector. A method for preparing a recombinant protein according to any one of claims 1 to 6, wherein the method is:
Introducing the construct according to any one of claims 11 to 14 into mammalian cells;
Culturing the mammalian cells under conditions suitable for protein expression and secretion to obtain the recombinant protein,
A method characterized by including. 15. The method of claim 15, wherein the mammalian cell is at least one selected from HEK1, CHOS, 293F, and 293T. A therapeutic composition for treating cancer, which comprises the recombinant protein according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. Use of the recombinant protein according to any one of claims 1 to 6 or a therapeutic composition containing the recombinant protein for producing a medicament for treating cancer in a patient suffering from cancer.

Images